我们感谢U.阿瑟瑞博士NPY-MRFP的cDNA的礼物。我们感谢博士。 MJ Kofron，L. Mittleman，M. Shaharbani和Y. Zilberstein与显微镜宝贵援助和图像分析。我们也感谢约瑟夫·奥利博士这个手稿的批判性阅读。这项工作是由以色列科学基金会的资助，由以色列科学院科学支持成立（1139至1112年，以RS-E）。

1.准备RBL细胞培养基<ol><li>混合500毫升低葡萄糖Dulbecco改进的Eagle培养基（DMEM）与56毫升胎牛血清（这使得10％FBS），然后添加5.5青霉素链霉素毫升（这使得〜1％PEST）。 </li><li>用500-1000毫升瓶顶真空过滤器具有0.22微米的孔径大小和存储在4℃过滤介质。 </li></ol> 2.文化RBL细胞<ol><li>在任一板或培养瓶生长RBL细胞在5％的CO 2的潮湿气氛中在37℃。 RBL细胞正在生长于10cm板应补充有10毫升培养基，形成的〜10 7个细胞/板汇合单层。 </li><li>当细胞层将至合流，replate培养以较低的密度。 <ol><li>除去从板/烧瓶用无菌巴斯德移液管连接到真空中的培养基中。 </li><li>冲洗细胞单层用预热（37℃）0.25％胰蛋白酶/ EDTA，涵盖了整个单层（体积取决于培养板/瓶的大小;2毫升为10cm平板）。这将分离的细胞。 </li><li>或者，冲洗细胞单层用磷酸盐缓冲盐水（PBS），除去PBS并加入预热（37℃）胰蛋白酶/ EDTA。这一步分离的细胞快。 </li><li>放置板/烧瓶在恒温箱在37℃（不超过10分钟）。 </li><li>检查该光学显微镜下的细胞，以检查它们是否已​​经脱离。如果细胞10分钟后没有脱落，轻轻拍打上盘/瓶。 </li><li>添加培养基向培养板/烧瓶以悬浮细胞（该介质的体积应至少2倍比胰蛋白酶溶液的体积大）。 </li><li>沉淀细胞离心3分钟，以200g在25℃。 </li><li>重悬在培养基中的细胞。由1:10稀释的细胞和种子在一个新的培养皿中。孵育细胞48-72小时，直到他们REACħ大约90％汇合。 </li><li>长达3个月（30代）重复相同的过程（部分2.2.1-2.2.8）。 </li></ol></li></ol> 3.准备转染媒体。 <ol><li>制备K -管道pH值7， 钙离子乙酸1mM溶液和镁离子醋酸100mM的溶液的100毫溶液。 </li><li>稀释的K-管道的pH 7的100mM的溶液成使用DMEM培养基20毫米， 钙醋酸1mM溶液添加至10微米，镁离子醋酸100mM的溶液的最终浓度为2的终浓度毫米。 </li><li>权衡谷氨酸钾，并添加到该溶液中，以128毫米的最终浓度，拌匀，并保持在4℃直至使用。保持其余的解决方案（ 即 ，K -管道， 钙离子，乙酸镁离子乙酸酯）于-20℃。 </li></ol> 4.转染RBL细胞<ol><li>从使用该板/烧瓶除去培养基无菌巴斯德吸管连接至真空。 </li><li>冲洗细胞单层用预热（37℃）胰蛋白酶/ EDTA应覆盖整个单层。放置板/烧瓶在恒温箱在37℃（不超过10分钟）。检查，如果细胞用光学显微镜已经分离。 </li><li>添加培养基向培养板/烧瓶以悬浮细胞（在介质的体积应至少2大于胰蛋白酶溶液的体积更大）。 </li><li>使用血细胞计数器计数细胞数。 </li><li>沉淀1.5×10 7个细胞离心3分钟，在200×g离心25℃。弃去上清液，加入280微升转介质。转移反应混合物成4mm杯。 </li><li>加入20 NPY-MRFP质粒微克，要么30微克控制空质粒或测试质粒。该反应混合物的最终体积应为300微升。 </li><li>立即放置在冰上10分钟。擦去残留的水和对反应杯roceed电穿孔在300V 9毫秒。 </li><li>为胞吐的测量，在含有300微升培养基的24孔组织培养皿中立即replate细胞。加入8微升的反应混合物（4×10 5细胞）至各孔。加入4×10 5个非转染细胞额外井控制。准备足够的孔中至少有两个孔，每个处理为每转染。 </li><li>对于时间推移显微镜，在含有80微升培养基的8孔腔室硼硅酸盐玻璃罩系统立即replate细胞。添加1.5微升的反应混合物（7.5×10 4个细胞）至各室。 （尽量避免使用该室在玻璃罩的边缘，该室在玻璃罩的中心更容易图像）。 注：重要的是，并非所有的细胞响应于触发和偶尔的时间推移显微镜是由于失去焦点的8孔腔室borosi和漂移废止licate玻璃罩系统。因此，准备至少8室，每个处理每个转染。 </li><li>对于敏化的细胞为了FcεRI介导的活化，加入小鼠DNP特异性单克隆IgE的1微克/毫升到介质（孵育的细胞的IgE至少2小时）。 </li><li>后18-24小时用荧光显微镜，以确认该细胞表达的质粒。 MRFP荧光的激发和发射波长分别是：λEx584，λEm607纳米。 NPY-MRFP应该出现在对应于SGS的囊泡结构。如果感兴趣的第二个基因融合到荧光标签使用荧光显微镜来确认的共表达两种质粒在相同的细胞。例如，如果所测试的基因融合到绿色荧光蛋白，GFP荧光的激发和发射波长分别是：λEx488，λEm507纳米。绿色荧光蛋白标记的蛋白质应出现在表达NPY-MRFP相同的细胞。 </li><li>对于胞外分泌的测量进行吨Ø第5步和时间的推移显微镜，继续执行步骤6。 </li></ol> 5.测量NPY-MRFP胞吐作用<ol><li>制备台氏缓冲液： <ol><li>制备的54毫米氯化钾，20mM的MgCl 2的，2.74 M氯化钠和8毫的NaH 2 PO 4在DDW中的溶液。拌匀，保存在4℃。本步骤是制备的20倍的Tyrode缓冲液中。 </li><li>制备的20毫摩尔的Hepes中的溶液pH值为7，1.8毫氯化钙 ，1毫克/毫升BSA，5.6毫葡萄糖和1至20的稀释20倍的Tyrode在DDW中并充分混合。本步骤是制备1X的Tyrode缓冲液中。 </li><li>分装1X的Tyrode缓冲液并储存在-20℃。避免反复冻融。 </li></ol></li><li>除去从24孔板的培养基中洗3次的Tyrode缓冲液中。通过加入200微升的Tyrode缓冲液以控制井制备未刺激细胞。 </li><li>制备10微升/孔20X的浓缩活化试剂〔（ 例如 1000纳克/毫升的DNP-BSA或DNP-HSA（银），200μM的Ca 2+离子载体（例如 A23187），和20微米的Ca 2+离子载体和1000纳米12-O-十四烷-13-乙酸酯（TPA）]的组合。如果试剂被储存在DMSO中，稀释试剂成20倍的浓度中含有1％DMSO中的Tyrode缓冲液中。孵育在37℃下30分钟。 </li><li>以及小心取出每个上清液至96孔板，放置在冰上，并避免光照。 （上清液包含所述嵌合肽的NPY-MRFP被从细胞中释放）。 </li><li>加含有0.5％的Triton X-100，以每200微升的Tyrode缓冲液，并培育在37℃下进行10分钟。 （本步骤是制备包含NPY-MRFP为没有从细胞中释放的剩余的细胞裂解物的重要）。收集细胞裂解物，并转移到一个96孔板中，放置在冰上，并避免光照。 </li><li>使用fluoresc测量细胞上清液和细胞裂解物的荧光ENCE板读数器，使用的是590-，20纳米的带宽激发滤光片和635-，35纳米的带宽发射滤光片。 </li><li>计算NPY-MRFP公布的百分比： 注：荧光读取器测量任意荧光单位（AFU）。 AFU值取决于机器和它的灵敏度和转染效率上。 <ol><li>设置未转染的RBL细胞作为空白的自体荧光。将每个上清液的总荧光的AFU（上清液+ AFU相应裂解物的AFU）再乘以100。 </li></ol></li></ol>胞吐6.定时显微镜<ol><li>在8孔室硼硅酸盐玻璃罩系统种子7.5×10 4个转染细胞/室。后18-24小时，从所述腔室取出培养基洗3次，用蒂罗德缓冲</li><li>加入72微升的Tyrode缓冲液到每个室中。淡化台氏缓冲的激活剂，以10倍的浓度。 </li> &lt;li&gt;使用配备有加热室（37°C）和CO 2控制器（4.8％）和40X或63X客观的共聚焦荧光显微镜。打开显微镜系统：汞灯，计算机，激光器。确保该加热室是在合适的温度在开始实验之前。 </li><li>将室中的加热室中，并确保该室被正确和稳定的安装。 </li><li>根据相关的荧光团开启荧光灯和可视化转染的细胞。关闭荧光一旦感兴趣的细胞是在现场，以尽量减少漂白和细胞毒性。这个字段将包含约2-3转染的细胞是重要的。转染的细胞应良好分散，但不互相接触。 </li><li>调整激光功率（取决于显微镜）以减少噪声和过饱和，以及毒性，并设置增益和偏移修改信噪比。在扫描速度快或德，以尽量减少激光博览会（平均2）的持续时间。 </li><li>调节针孔尺寸到最大，这使得能够降低激光功率，并保持集中的时间很长的周期的图像。如果需要的话，设置参数为Z-堆栈来重建图像在三维，调整针孔大小为1通风单元（AU），该给出最佳的信噪比，并获得连续的二维共焦光学切片扫描ž-axes光学切片≤0.7微米。 </li><li>设定每个采集到15-30秒，总采集到15分钟的持续时间之间的间隔时间，并开始获取图像。 5分钟后，采集的暂停时间序列。添加8微升10倍的触发，并立即继续进行收购。 </li><li>保存的图片，采用了卷积软件进行反褶积和重​​建书库三维图像，并使用了Imaris软件的一部电影。 </li></ol><ol><li>从共聚焦荧光显微镜来了Imaris软件获得的解卷积时间序列图像导入数据。该软件可读取超过40显微镜文件。如果软件无法读取的文件;将文件转换为TIF文件和进口。 </li><li>时间点添加到一个开放的数据集新闻编辑结束 - &gt;添加时间点，并导入新的数据集。 </li><li>创建使用表面向导选项MRFP通道的表面。选择默认的算法。选择的NPY-MRFP的信道。纪念平滑选项，以减少噪音。为了避免损失的小细节降低区域的细节层面，以0.05微米。 </li><li>设定强度阈值。新的灰面将显示。进入“设置”，并从“中心点”到“面”切换风格。 </li><li>转到所选对象的属性的统计选项卡中，选择详细的标签和特定值SUCH作为荧光强度，音量等。 </li><li>检查数据并确认值与图像相兼容。例如，两个或更多个相邻的颗粒可能被测量为1大颗粒。用于量化的平均颗粒尺寸除以合并的颗粒，以颗粒剂的实际数量的大小。 </li><li>导出所需的数据集到Excel文件和分析数据。 </li></ol> ˚F

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P., Matsui, T., Fukuda, M., Sagi-Eisenberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+regulation+of+mast+cell+exocytosis+by+networks+of+Rab+GTPases.">Decoding the regulation of mast cell exocytosis by networks of Rab GTPases.</a> J Immunol. 189 (5), 2169-2180 (2012).</li><li>Stenmark, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+rab5+GTPase+activity+stimulates+membrane+fusion+in+endocytosis.">Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.</a> EMBO J. 13 (6), 1287-1296 (1994).</li><li>Azouz, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5+is+a+novel+regulator+of+mast+cell+secretory+granules:+impact+on+size,+cargo,+and+exocytosis.">Rab5 is a novel regulator of mast cell secretory granules: impact on size, cargo, and exocytosis.</a> J Immunol. 192 (9), 4043-4053 (2014).</li></ol>

The authors have no financial conflicts of interest.

我们描述了一种创新的策略，通过时间流逝立体使用报道基因用于胞吐作用在活细胞中的SG成像结合的MC胞吐和4（X，Y，Z，t）的维数的量化值的量化。这项技术使蛋白质他们对MC功能影响的家庭，如监测的SG，早在他们从高尔基通过其成熟的退出，收购胞外分泌能力和脱粒开始筛选。胞吐连同了SGS的形态和动能表征测量相结合，提供了深入了解通过该测试的蛋白介导MC功能的机制。因此，使用这种?...

肥大细胞（MC）是最知名的是它们在过敏性和炎性反应，如关节炎，哮喘，嗜酸细胞性食管炎，慢性皮炎和过敏性休克1,2-以及其他疾病，包括冠状动脉疾病3,5-和参与免疫细胞癌症3,4。此外，管委会发挥固有免疫和适应性免疫的重要角色，无论是在对抗细菌和寄生虫的宿主防御和抑制免疫反应，例如诱导移植耐受5,6。 的MC从骨髓来源，来自CD34 + / CD117 +多潜能祖细胞7显影。致力于骨髓祖细胞的MC被释放到血液中，并迁移到结缔组织和上皮表面8内定位主要的外周组织。成熟和终末分化的影响o在最终会实现f显示周围环境8,9内细胞因子。 管委会可通过过敏原（抗原，抗原），其遭遇导致免疫球蛋白E（IgE）的一代型抗体被激活。这样的IgE结合到MC的了FcεRI受体，随后交联的细胞结合的IgE的经重新暴露于相同的银，导致了FcεRI聚集和一个信号级联高潮在细胞脱粒的起始[10,11中综述] 。的MC也被激活，独立的IgE，通过神经肽5,12，毒素13，细菌和病毒抗原14,15，多个带正电荷的肽统称为基本分泌素，免疫细胞和细胞因子5,13,12,16的17。的MC是由许多自身释放介质，其进一步放大炎性反应也被激活。 管委会都挤满了分泌颗粒（SGS）含有免疫调节介质，包括血管活性胺，如组胺和血清素（在啮齿动物），蛋白聚糖，蛋白酶，如胃促胰酶和类胰蛋白酶，血管内皮生长因子和多种细胞因子和趋化因子8,9。这些介质是“准备好”，并一次的MC是由一个适当的刺激物激活，这些介质被从细胞通过在几秒钟到几分钟18,19事项调节胞吐作用（脱粒）释放。这个初始事件之后是从头合成一个大阵列的生物效物质，包括花生四烯酸代谢物，多种细胞因子和趋化因子20,21,22和释放。新合成的产品发布时的独立SG发布。总的来说，这些调解员开展早期和晚期炎症和过敏性反应。因此，了解占MC激活和脱颗粒的机制是理论和临床小鬼ortance。 转基因操纵初级和培养管委会的困难阻碍了试图阐明基本MC脱颗粒的机制，仍然解决不好。为了克服这个问题，我们开发了一种基于记者通过测定共转染的粘膜肥大细胞系，大鼠嗜碱性白血病（RBL）-2H3（本文中称为RBL）或骨髓衍生的MC（的BMMCs）30与感兴趣的基因和神经肽Y（NPY）融合到单体RFP（MRFP），作为SG记者。 NPY先前所示扼要重述在其它系统的内源性SG标志物的行为。此外，由于荧光MRFP是pH值不敏感的，表达NPY-MRFP允许酸性的SG的可视化以及胞吐作用的定量评估通过使用96孔板和荧光板读数器。我们已经表明，NPY-MRFP被输送到RBL细胞和的BMMCs的酸性SGS和从该细胞释放在经调节的方式沿着内源性SG货物（ 即 ，β氨基己糖苷酶和血清素）30，32。这个协议提供了高分辨率成像为基础的方法，它允许对他们的表型和在RBL细胞32上SG上的特点和脱粒功能影响兴趣基因的筛选。具体地，该协议允许的MC的SG的实时跟踪和它们的面积或体积的大小，它们的数量，组装的动力学，其沿着细胞骨架的运动以及它们在不同条件下质膜最终融合的定量。例如，用DNP特异性IgE致敏的细胞和触发细胞用多价抗原（DNP缀合的血清白蛋白）根据不同的扰动（ 即 ，击倒的感兴趣的基因，过的重量或突变基因的表达，或药理学操作），并比较，以控制细胞。

<table border="0" cellpadding="0" cellspacing="0" width="844">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Name of Material/ Equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:156px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:421px;">DMEM</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D6046-500ML</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Fetal Bovine Serum</td>
			<td style="width:128px;">GE health care Life sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td style="width:128px;">Life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Cellulose acetate membrane, pore size 0.22&#160;&#956;m</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430769-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning tissue-culture treated culture dishes</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:128px;">Life technologies</td>
			<td>R001100</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">PIPES dipotassium salt</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">108321-27-3&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium acetate hydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>114460-21-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium acetate tetrahydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">M5661&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">L-Glutamic acid potassium salt monohydrate (Potassium glutamate)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 mm electroporation cuvettes</td>
			<td style="width:128px;">cell projects</td>
			<td style="width:139px;">EP-104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GENE PULSER WITH PULSE CONTROLLER &#38; CAPACITANCE</td>
			<td style="width:128px;">Bio rad</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chambered coverglass</td>
			<td style="width:128px;">Thermo scientific</td>
			<td align="right" style="width:139px;">155411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">24 well, flat bottom</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Corning 96 well plates</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3367 or CLS390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate fluorescence reader- Infinite 200</td>
			<td style="width:128px;">Tecan</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium ionophore A23187</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">C7522</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-O-tetradecanoyl-13-acetate (TPA)</td>
			<td>Calbiochem</td>
			<td height="20" style="height:20px;width:139px;">P3766</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-DNP monoclonal IgE</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D8406&#160;</td>
			<td height="20" style="height:20px;width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNP-BSA/ DNP-HAS</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A6661</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Triton-x-100</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Confocal fluorescent microscope:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Zeiss LSM 510</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Leica</td>
			<td style="width:128px;">SP5</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon A1 inverted</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Imaris software</td>
			<td style="width:128px;">BITLANE</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Microsoft exel or Prism or other analyses software</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Other reagent:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium Chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5833</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sodium chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">6404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium chloride&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">2382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A4503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Glucose</td>
			<td style="width:128px;">BDH Laboratories</td>
			<td style="width:139px;">284515V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Monosodium phosphate&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sterile water</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating mast cell secretory granules; from biosynthesis to exocytosis

肥大细胞（MC）是免疫系统的分泌细胞完成其生理和病理功能通过释放预先形成和新合成的过敏性，炎性和免疫调节介质。 MC的中介影响多个组织和器官最终在过敏和免疫反应。在MC介质的合成，储存和释放受到严格的监管。预形成的介质都装在胞质分泌粒剂（SG），该融合与质膜和由​​稳压胞吐作用释放其内容。我们提出了一个协议的基础上，共表达的与一个报告基因的兴趣被靶向SGS和被释放的调节的方式沿着内源性SG介质的基因。该协议使高分辨率四维共聚焦分析了MC的SGS和监控，从生源的时间表，以触发胞吐。因此，使用该协议除筛选基因EST他们的表型和功能的影响允许破译支配的MC的SG的生物合成和胞吐作用的分子机制，并确定所涉及的监管机构。由此，进一步的见解认为占在健康和疾病的MC功能的细胞机制应提供。

因为市政局的转染效率低的，遗传操作是不可能离开平均分泌的内源性的SG介质测量读数产生影响。然而，由在监测只表达感兴趣的基因的细胞群体建立完整的共表达的报告基因的NPY-MRFP和共转染质粒在相同细胞中，监测的NPY-MRFP结果。因此，这种测定法与传统方法相比的优点是能够选择性地监测遗传操作的细胞（ 图1）的能力。 事实上，采用此法，我们筛选基因?...

Watch this Scientific Journal Video about Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis at JoVE.com

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

本议定书的目标是开发一种方法，使肥大细胞分泌功能基因组分析。所述协议是基于荧光报道基因cotrasfected与感兴趣和实时的基因的释放定量评估分析分泌颗粒的形态。

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

investigating-mast-cell-secretory-granules-from-biosynthesis-to

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JoVE Journal

Immunology and Infection

13.0K 次观看.  Sackler Faculty of Medicine, Tel Aviv University. 本议定书的目标是开发一种方法，使肥大细胞分泌功能基因组分析。所述协议是基于荧光报道基因cotrasfected与感兴趣和实时的基因的释放定量评估分析分泌颗粒的形态。 

视频: Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

科研

免疫与感染

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the &#8216;mast cell knock-in&#8217; approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

Analyzing the Functions of Mast Cells In Vivo Using &#039;Mast Cell Knock-in&#039; Mice

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

使用"桅杆细胞敲击"小鼠分析体内桅杆细胞的功能

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

通过使用来自辐照的Buffy外套的饲养细胞的T淋巴细胞的分离和培养来研究主动脉瓣钙化

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

使用来自同一物种的抗体进行双标记免疫荧光研究宿主 - 病原体相互作用

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.